Description
CDC2L1 Colorimetric Cell-Based ELISA
The CDC2L1 Colorimetric Cell-Based ELISA Kit is a powerful tool for analyzing the activity of CDC2L1 in cell-based assays. This kit provides a simple and efficient method for quantifying CDC2L1 levels in cell lysates, allowing researchers to study the role of this protein in various cellular processes.CDC2L1 is a key regulator of cell cycle progression and has been implicated in the development of cancer and other diseases. By using the CDC2L1 Colorimetric Cell-Based ELISA Kit, researchers can gain valuable insights into the function of CDC2L1 and its potential as a therapeutic target.
With its high sensitivity and specificity, the CDC2L1 Colorimetric Cell-Based ELISA Kit offers reliable and reproducible results, making it an essential tool for researchers looking to advance their understanding of CDC2L1 biology. Upgrade your research capabilities with this innovative kit today.
| Product Name: | CDC2L1 Colorimetric Cell-Based ELISA |
| Product Code: | CBCAB00986 |
| ELISA Type: | Cell-Based |
| Target: | CDC2L1 |
| Reactivity: | Human, Mouse |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The CDC2L1 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect CDC2L1 protein expression profile in cells. The kit can be used for measuring the relative amounts of CDC2L1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on CDC2L1.
Qualitative determination of CDC2L1 concentration is achieved by an indirect ELISA format. In essence, CDC2L1 is captured by CDC2L1-specific primary antibodies while the HRP-conjugated secondary antibodies bind the Fc region of the primary antibody. Through this binding, the HRP enzyme conjugated to the secondary antibody can catalyze a colorimetric reaction upon substrate addition. Due to the qualitative nature of the Cell-Based ELISA, multiple normalization methods are needed:
| 1. | A monoclonal antibody specific for human GAPDH is included to serve as an internal positive control in normalizing the target absorbance values. |
| 2. | Following the colorimetric measurement of HRP activity via substrate addition, the Crystal Violet whole-cell staining method may be used to determine cell density. After staining, the results can be analysed by normalizing the absorbance values to cell amounts, by which the plating difference can be adjusted. |
| Database Information: | Gene ID: 984, UniProt ID: P21127, OMIM: 176873, Unigene: Hs.651228/Hs.709182 |
| Gene Symbol: | CDK11B |
| Sub Type: | None |
| UniProt Protein Function: | Plays multiple roles in cell cycle progression, cytokinesis and apoptosis. Involved in pre-mRNA splicing in a kinase activity-dependent manner. Isoform 7 may act as a negative regulator of normal cell cycle progression. |
| NCBI Summary: | This gene encodes a member of the serine/threonine protein kinase family. Members of this kinase family are known to be essential for eukaryotic cell cycle control. Due to a segmental duplication, this gene shares very high sequence identity with a neighboring gene. These two genes are frequently deleted or altered in neuroblastoma. The protein kinase encoded by this gene can be cleaved by caspases and may play a role in cell apoptosis. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Apr 2014] |
| UniProt Code: | P21127 |
| NCBI GenInfo Identifier: | 34978359 |
| NCBI Gene ID: | 984 |
| NCBI Accession: | P21127.3 |
| UniProt Secondary Accession: | P21127,O95265, Q12817, Q12818, Q12819, Q12820, Q12822 Q8N530, Q9NZS5, Q9UBJ0, Q9UBQ1, Q9UBR0, |
| UniProt Related Accession: | P21127 |
| Molecular Weight: | 49,555 Da |
| NCBI Full Name: | Cyclin-dependent kinase 11B |
| NCBI Synonym Full Names: | cyclin-dependent kinase 11B |
| NCBI Official Symbol: | CDK11B |
| NCBI Official Synonym Symbols: | p58; PK58; CDK11; CLK-1; CDC2L1; PITSLREA; p58CLK-1; CDK11-p46; CDK11-p58; p58CDC2L1; CDK11-p110 |
| NCBI Protein Information: | cyclin-dependent kinase 11B |
| UniProt Protein Name: | Cyclin-dependent kinase 11B |
| UniProt Synonym Protein Names: | Cell division cycle 2-like protein kinase 1; CLK-1; Cell division protein kinase 11B; Galactosyltransferase-associated protein kinase p58/GTA; PITSLRE serine/threonine-protein kinase CDC2L1; p58 CLK-1 |
| Protein Family: | Cyclin-dependent kinase |
| UniProt Gene Name: | CDK11B |
| UniProt Entry Name: | CD11B_HUMAN |
| Component | Quantity |
| 96-Well Cell Culture Clear-Bottom Microplate | 2 plates |
| 10X TBS | 24 mL |
| Quenching Buffer | 24 mL |
| Blocking Buffer | 50 mL |
| 15X Wash Buffer | 50 mL |
| Primary Antibody Diluent | 12 mL |
| 100x Anti-Phospho Target Antibody | 60 µL |
| 100x Anti-Target Antibody | 60 µL |
| Anti-GAPDH Antibody | 60 µL |
| HRP-Conjugated Anti-Rabbit IgG Antibody | 12 mL |
| HRP-Conjugated Anti-Mouse IgG Antibody | 12 mL |
| SDS Solution | 12 mL |
| Stop Solution | 24 mL |
| Ready-to-Use Substrate | 12 mL |
| Crystal Violet Solution | 12 mL |
| Adhesive Plate Seals | 2 seals |
The following materials and/or equipment are NOT provided in this kit but are necessary to successfully conduct the experiment:
- Microplate reader able to measure absorbance at 450 nm and/or 595 nm for Crystal Violet Cell Staining (Optional)
- Micropipettes with capability of measuring volumes ranging from 1 µL to 1 ml
- 37% formaldehyde (Sigma Cat# F-8775) or formaldehyde from other sources
- Squirt bottle, manifold dispenser, multichannel pipette reservoir or automated microplate washer
- Graph paper or computer software capable of generating or displaying logarithmic functions
- Absorbent papers or vacuum aspirator
- Test tubes or microfuge tubes capable of storing ≥1 ml
- Poly-L-Lysine (Sigma Cat# P4832 for suspension cells)
- Orbital shaker (optional)
- Deionized or sterile water
*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
| Step | Procedure |
| 1. | Seed 200 µL of 20,000 adherent cells in culture medium in each well of a 96-well plate. The plates included in the kit are sterile and treated for cell culture. For suspension cells and loosely attached cells, coat the plates with 100 µL of 10 µg/ml Poly-L-Lysine (not included) to each well of a 96-well plate for 30 minutes at 37°C prior to adding cells. |
| 2. | Incubate the cells for overnight at 37°C, 5% CO2. |
| 3. | Treat the cells as desired. |
| 4. | Remove the cell culture medium and rinse with 200 µL of 1x TBS, twice. |
| 5. | Fix the cells by incubating with 100 µL of Fixing Solution for 20 minutes at room temperature. The 4% formaldehyde is used for adherent cells and 8% formaldehyde is used for suspension cells and loosely attached cells. |
| 6. | Remove the Fixing Solution and wash the plate 3 times with 200 µL 1x Wash Buffer for five minutes each time with gentle shaking on the orbital shaker. The plate can be stored at 4°C for a week. |
| 7. | Add 100 µL of Quenching Buffer and incubate for 20 minutes at room temperature. |
| 8. | Wash the plate 3 times with 1x Wash Buffer for 5 minutes each time. |
| 9. | Add 200 µL of Blocking Buffer and incubate for 1 hour at room temperature. |
| 10. | Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time. |
| 11. | Add 50 µL of 1x primary antibodies (Anti-CDC2L1 Antibody and/or Anti-GAPDH Antibody) to the corresponding wells, cover with Parafilm and incubate for 16 hours (overnight) at 4°C. If the target expression is known to be high, incubate for 2 hours at room temperature. |
| 12. | Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time. |
| 13. | Add 50 µL of 1x secondary antibodies (HRP-Conjugated AntiRabbit IgG Antibody or HRP-Conjugated Anti-Mouse IgG Antibody) to corresponding wells and incubate for 1.5 hours at room temperature. |
| 14. | Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time. |
| 15. | Add 50 µL of Ready-to-Use Substrate to each well and incubate for 30 minutes at room temperature in the dark. |
| 16. | Add 50 µL of Stop Solution to each well and read OD at 450 nm immediately using the microplate reader. |
(Additional Crystal Violet staining may be performed if desired – details of this may be found in the kit technical manual.)
